Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.

Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3′UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.